Transplantation of islets of Langerhans holds great promise to improve treatment of Type 1 diabetes mellitus, but the scarcity of available donor islets is one obstacle that need to be addressed. Pluripotent stem cells can in principle generate unlimited numbers of beta cells for transplantation but reliable protocols for generating fully functional beta cells are not yet developed. The foregut derivatives; pancreas, lung, thyroid, liver, esophagus, and stomach originate from definitive endoderm (DE), one of the three germ layers that forms during gastrulation. Induction of DE is the first critical step towards formation of differentiated cell types from endodermally derived tissues such as insulin producing beta cells of the pancreas. Formation of DE cells from pluripotent embryonic stem (ES) cells has been reported for both mouse and human in, e.g., WO2005/116073, WO2005/063971, and US 2006/0148081. The generation of pancreatic endoderm (PE) cells from DE cells is necessary for the generation of insulin-producing beta cells for the treatment of diabetes.
It is known that DE formation in the embryo passes through the intermediate step of primitive streak (PS) formation (a mesendodermal intermediate) which has the potential to form either mesoderm or DE. At the cellular level, all developmental processes are ultimately controlled by the cooperative actions of different signal transduction pathways. Among them, Wnt signaling is indispensable for orchestrating the complex cell behaviors that occur throughout development. Wnt signaling controls cell proliferation, stem cell maintenance and cell fate decisions, as well as organized cell movements and the establishment of tissue polarity. It is also frequently deregulated in human cancers and has been implicated in degenerative diseases. As a potential target for therapeutic intervention, it thus holds new promises in the fields of stem cell biology and regenerative medicine.
Induction of DE has been described by the use of Activin A/Nodal with or without the combination of Wnt. In particular, the Wnt receptor ligand Wnt3a has previously been reported to contribute to efficient DE formation when incubated along with activin A (AA) during day 1 (24 h) of a 5 day AA-mediated induction of DE, ie., using the conventional D'Amour protocol (Kroon et al 2008, Nat Biotech, 2006).
Attempts to group individual Wnt proteins into classes to which specific activities could be assigned have resulted in the subdivision of Wnts into ‘canonical’ or ‘non-canonical’, based on the ability of the former, but not the later, to induce beta-catenin/TCF signaling.
CHIR is a glycogen synthase kinase 3 beta (Gsk3b) inhibitor and a known component of a defined tissue culture medium to maintain mouse embryonic stem cells in the pluripotent state (Ying et al Nature 453, 519-523). Gsk3b has multiple targets but is mainly known to regulate degradation and/or nuclear transfer of beta-catenin. The role of stabilized beta-catenin in PS formation from human embryonic stem cells (hESC) has been described using other glycogen synthase kinase 3 beta (Gsk3b) inhibitors such as BIO and Wnt3a. However CHIR is the most selective Gsk3b-inhibitor reported to date.
Co-incubation of AA and CHIR reduces DE formation stability, leading to a less efficient and less robust protocol. The present invention relates to the utilization of CHIR without AA, at a defined concentration range to treat pluripotent stem cells prior to AA-mediated definitive endoderm induction compared to the conventional induction protocol for obtaining DE (D'Amour protocol, as described in Kroon et al. 2008). This sequential exposure, first to CHIR and then to AA, is essential and reflects successive PS formation (induced by CHIR) followed by more efficient and rapid DE formation by AA. The present invention relates to the discrete and successive control of induction of first PS then followed by DE leading to an overall more efficient, with earlier peak of SOX17 expression and robust DE protocol. These effects of CHIR cannot be reproduced with Wnt3a treatment.